The objective of this proposal is to explore the relationship of the structure of glucagon to its biological function. The mechanism of action of this hormone must be understood to define its role in the normal individual and in the diabetic patient. Taking advantage of the sole methionine of glucagon which can be reversibly methylated without damage to the molecule, we have prepared a number of highly purified, well-characterized derivatives of the carboxyl-terminal region of the hormone. Those already prepared are currently being assessed for their helix-forming potential, their binding to rat liver plasma membranes and their activation of adenyl cyclase. We will extend the number and variety of derivatives to include those obtained by further enzymatic and chemical cleavage reactions. By methods of semisynthesis we expect to couple synthetically prepared and protected peptides to the amino terminus of peptide fragments derived from native hormone to form synthetic hybrid hormones containing the carboxyl-terminal portion of glucagon and the amino-terminal portion of evolutionarily related peptides. These will be useful in evaluating the requirements for activation of adenyl cyclase as well as those for binding. We further will trace the internalization of glucagon using the Protein-A-colloidal gold method to visualize immunoreactive glucagon within the cell by electron microscopy. If immunoreactive glucagon is internalized, subcellular fractionation will be used to isolate purified organelles whose binding of glucagon and its derivatives will also be characterized. We will particularly be searching for a glucagon antagonist which could be useful for in vivo studies of the effects of glucagon in experimental models of diabetes mellitus.